WO2003080817A1 - Procede de production de lymphocyte cytotoxique - Google Patents

Procede de production de lymphocyte cytotoxique Download PDF

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Publication number
WO2003080817A1
WO2003080817A1 PCT/JP2003/003575 JP0303575W WO03080817A1 WO 2003080817 A1 WO2003080817 A1 WO 2003080817A1 JP 0303575 W JP0303575 W JP 0303575W WO 03080817 A1 WO03080817 A1 WO 03080817A1
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Prior art keywords
fibronectin
cells
culture
fragment
polypeptide
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PCT/JP2003/003575
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English (en)
Japanese (ja)
Inventor
Hiroaki Sagawa
Mitsuko Ideno
Ikunoshin Kato
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Takara Bio Inc.
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Publication date
Priority to US10/509,055 priority Critical patent/US8728811B2/en
Priority to AT03712887T priority patent/ATE491021T1/de
Priority to CA2479288A priority patent/CA2479288C/fr
Priority to KR1020047014928A priority patent/KR100786054B1/ko
Application filed by Takara Bio Inc. filed Critical Takara Bio Inc.
Priority to JP2003578546A priority patent/JP4406566B2/ja
Priority to AU2003221073A priority patent/AU2003221073B2/en
Priority to EP03712887A priority patent/EP1496109B1/fr
Priority to MXPA04009287A priority patent/MXPA04009287A/es
Priority to DE60335253T priority patent/DE60335253D1/de
Priority to EA200401242A priority patent/EA010434B1/ru
Publication of WO2003080817A1 publication Critical patent/WO2003080817A1/fr
Priority to HK05111402.4A priority patent/HK1079543A1/xx
Priority to US11/831,423 priority patent/US20080227204A1/en
Priority to AU2008243221A priority patent/AU2008243221B2/en
Priority to US14/250,227 priority patent/US8975070B2/en

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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
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    • A61K39/4613—Natural-killer cells [NK or NK-T]
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12N2501/50—Cell markers; Cell surface determinants
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a method for obtaining cytotoxic lymphocytes useful in the medical field.
  • lymphocytes Living organisms are protected from foreign substances mainly by immune responses, and the immune system is made up of various cells and the soluble factors they produce. Among them, leukocytes, especially lymphocytes, play a central role. These lymphocytes are divided into two main types: B lymphocytes (hereinafter sometimes referred to as B cells) and T lymphocytes (hereinafter sometimes referred to as T cells). It recognizes antigens specifically and acts on them to protect the body.
  • B lymphocytes hereinafter sometimes referred to as B cells
  • T lymphocytes hereinafter sometimes referred to as T cells
  • CD Crat ter Des ignat ion
  • T H helper T cells involved in the induction of antibody production aid and various immune responses
  • CD 8 markers are cytotoxic T lymphocytes (cyto toxic T ly immediately Hocyte), also known as killer one T cell; have one, predominantly cytotoxic T cells that exhibit cytotoxic activity [T C. Hereinafter, it may be described as CTL). Recognize and destroy tumor cells and virus-infected cells, etc. The most important role is to produce antibodies that react specifically with antigens, such as sputum cells.
  • HLA human leukocyte antigen
  • Target cells determined to be non-self-derived are specifically destroyed and removed by CTL.
  • TIL tumor-infiltrating lymphocytes
  • Rosenberg SA Rosenberg SA, et al. (Rosenberg SA, et al.), N. Engl. J. Med., 319, 25, 1676-1680 (1988); Ho M., et al., Blood, 81, No. 8, pp. 2093-2101 (1993)] is known.
  • the WO 96/06929 pamphlet discloses the REM method (rapid expansion method).
  • This REM method is an antigen-specific C proliferate TL and T H in a short period of time the initial population of including T cell methods of (Exp and) is.
  • individual T cell clones can be expanded to provide a large amount of T cells, and anti-CD3 antibody, IL-12, and PBMC (peripheral blood mononuclear cell, peripheral blood mononuclear cell) that has been made non-proliferative by irradiation. It is characterized by increasing the number of antigen-specific CTL using nuclear cells) and Epstein-Barr virus (hereinafter abbreviated as EBV) infected cells.
  • EBV Epstein-Barr virus
  • WO 97/32970 pamphlet discloses a modified REM method, which uses a non-dividing mammalian cell line expressing a T cell stimulating component that is distinct from PBMC as a feeder cell.
  • Lymphokine-activated killer cells LAK cells
  • LAK cells a method of reducing the amount of PBMC used, add IL-12 to peripheral blood (peripheral blood leukocytes), umbilical cord blood, tissue fluid, etc. It is a functional cell population having cytotoxic activity obtained by culturing in vitro in a day.
  • peripheral blood peripheral blood leukocytes
  • umbilical cord blood tissue fluid
  • tissue fluid etc.
  • anti-CD3 antibody and culturing the proliferation of LAK cells is further accelerated.
  • the LAK cells thus obtained have non-specific cytotoxic activity against various cancer cells and other targets.
  • LAK cells are also used for adoptive immunotherapy as
  • IL-2 is indispensable in the process of obtaining cytotoxic lymphocytes such as CTL, LAK cells, TIL and the like.
  • Cells are further activated by IL-2 binding to the interleukin-2 receptor (IL-2R) on the cell surface.
  • IL-2R interleukin-2 receptor
  • IL-12R is known as a lymphocyte activation marker.
  • it is important to increase the expression of IL-2R on the cell surface.
  • the efficiency of inducing CTL progenitor cells subjected to stimulation with antigen as CTL is improved, that is, the ratio (ratio) of CD 8 positive cells in the cell group after induction is improved. It is important to do this.
  • Fibronectin is a huge glycoprotein with a molecular weight of 250,000 that is present in animal blood, on the surface of cultured cells, and in the extracellular matrix of tissues, and is known to have a variety of functions.
  • the domain structure is divided into seven parts (see Fig. 1 below), and the amino acid sequence contains three types of similar sequences. ing. Three types of similar sequences are called type I, type II, and type III. Of these, type III is composed of 71 to 96 amino acid residues, and the concordance rate of these amino acid residues. Is 17-40%. There are 14.
  • Type III sequences in fibronectin of which the 8th, 9th, and 10th (hereinafter referred to as III-8, 1 1 1-9, and III-10, respectively).
  • the 12th, 13th, and 14th cells are contained in the heparin binding domain.
  • III-10 also contains a VL A (verylateactivati on an tigen) -5 binding region,
  • the core sequence is RGDS.
  • I ⁇ ICS exists on the C-terminal side of the heparin-binding domain.
  • I ⁇ I CS has a region called CS-1, which has a binding activity to VL-4, which consists of 25 amino acids.
  • An object of the present invention is to provide a method for obtaining cytotoxic lymphocytes having high cytotoxic activity and suitable for medical use.
  • Cytotoxic lymph comprising the step of performing at least one of induction, maintenance and expansion of cytotoxic lymphocytes in the presence of fibronectin, a fragment thereof or a mixture thereof Sphere manufacturing method,
  • Cytotoxic lymphocytes are more cytotoxic than those induced and / or maintained in the absence of fibronectin, fragments thereof or mixtures thereof.
  • the fibronectin fragment is a polypeptide comprising at least one amino acid sequence represented by SEQ ID NOs: 1 to 7 in the sequence listing, or substitution of one or more amino acids in the amino acid sequence of the polypeptide, The method according to any one of [1] to [8], wherein the polypeptide has an amino acid sequence having a deletion, insertion, or addition, and has the same function as the polypeptide.
  • fibronectin fragment is a polypeptide selected from a polypeptide consisting of any one of the amino acid sequences represented by SEQ ID NOs: 8 to 19 in the Sequence Listing.
  • the ratio of the number of cells at the start of culture and the culture area in the cell culture equipment is 1 to 5 X 10 5 eel 1 s cm 2 ;
  • a medicament comprising cytotoxic lymphocytes obtained by the method according to any one of [1] to [13] as an active ingredient
  • the fibronectin fragment is a polypeptide comprising at least one amino acid sequence represented by SEQ ID NOs: 1 to 7 in the sequence listing, or substitution of one or more amino acids in the amino acid sequence of the polypeptide
  • An agent for improving the ratio of CD8-positive cells in lymphocytes comprising fibronectin, a fragment thereof or a mixture thereof as an active ingredient,
  • the fibronectin fragment is represented by SEQ ID NO: 1 to 7 in the sequence listing. Or a polypeptide having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions to the amino acid sequence of the polypeptide.
  • the ratio improving agent for CD8 positive cells according to [20] which is a polypeptide having a function equivalent to that of the polypeptide,
  • An agent for improving or maintaining cytotoxic activity in cytotoxic lymphocytes comprising fibronectin, a fragment thereof or a mixture thereof as an active ingredient,
  • the fibronegutine fragment is a polypeptide comprising at least one amino acid sequence represented by SEQ ID NOs: 1 to 7 in the sequence listing, or substitution of one or more amino acids in the amino acid sequence of the polypeptide
  • Improver or maintainer [28] Interleukins in cytotoxic lymphocytes comprising the step of inducing, maintaining and expanding cytotoxic lymphocytes in the presence of fibronectin, a fragment thereof or a mixture thereof.
  • CD8-positive cells in cytotoxic lymphocytes comprising the steps of induction, maintenance and expansion of cytotoxic lymphocytes in the presence of fibronectin, a fragment thereof or a mixture thereof. How to improve the ratio,.
  • a cell in a cytotoxic lymphocyte comprising the step of performing at least one of induction, maintenance and expansion of cytotoxic lymphocytes in the presence of fibronectin, a fragment thereof or a mixture thereof.
  • a method for improving or maintaining injury activity comprising the step of performing at least one of induction, maintenance and expansion of cytotoxic lymphocytes in the presence of fibronectin, a fragment thereof or a mixture thereof.
  • FIG. 1 is a schematic diagram showing the domain structure of fibronectin. BEST MODE FOR CARRYING OUT THE INVENTION
  • the present invention maintains high cytotoxic activity, significantly increases the expression level of IL-12R, and CD8 positive cells The ratio has been found to improve and have been completed.
  • the production of cytotoxic lymphocytes refers to a process including each step of induction (activation), maintenance, and expansion of the cell, or a combination of these steps.
  • the production of cytotoxic lymphocytes of the present invention is also referred to as culture of cytotoxic lymphocytes.
  • Fibronectin and fragments thereof used in the present invention may be obtained from nature or artificially synthesized. Fibronectin and its fragments are described in, for example, Ruoslahti E. et al. (Ruos laht i E., et al., Journal of Biochemistry. J. Biol. Chem. 7277-7281 (1981)], it can be produced in substantially pure form from naturally occurring substances.
  • the substantially pure fibronectin or fibronectin fragment described herein means that they are essentially free of other proteins that are naturally present with fibronectin.
  • Each of the above fibronectin and the fragment thereof can be used in the present invention alone or in a mixture of plural kinds.
  • fibronectin fragments that can be used in the present invention, as well as the preparation of such fragments, can be found in Kimika F. et al. [Kimiduka R, et al., Journal of Biochemistry. 284-291 (1991)], Cornbritt A. R. et al. [Kornbr iht t AR, et al., EMBO J.), Vol. 4, No. 7, 1755-1759 (1985) And Sekiguchi K., et al., Biochemistry, Vol. 25, No. 17, 4936-4941 (1986)], and the like.
  • fibronectin fragments include, for example, at least II 1-8 (amino acid sequence represented by SEQ ID NO: 1 in the sequence listing), III-1 9 ( Amino acid sequence represented by SEQ ID NO: 2 in the sequence listing), 1 1 1-10 (amino acid sequence represented by SEQ ID NO: 3 in the sequence listing), II 1-12 (expressed by SEQ ID NO: 4 in the sequence listing) Amino acid sequence), II 1-13 (amino acid sequence represented by SEQ ID NO: 5 in the sequence listing), II 1-14 (amino acid sequence represented by SEQ ID NO: 6 in the sequence listing), and CS-1 (sequence listing)
  • a polypeptide (see FIG. 1) comprising an amino acid sequence constituting any region of the amino acid sequence represented by SEQ ID NO: 7 is exemplified.
  • the fragment those having cell adhesion activity and / or heparin binding activity can be preferably used.
  • the cell adhesion activity can be examined by assaying the binding between the fragment used in the present invention (its cell binding domain) and the cell using a known method.
  • such methods include Williams D. A. et al. [Williams D. A., et al., Nature, Vol. 352, pp. 438-441 (1991)].
  • This method is a method for measuring the binding of cells to a fragment immobilized on a culture plate.
  • the heparin binding activity can be examined by assessing the binding of the fragment used in the present invention (its heparin binding domain) to heparin using a known method.
  • Heparin instead of cells, for example, heparin for labeling can be used to evaluate the binding of fragment to heparin in the same manner.
  • C-274 amino acid sequence represented by SEQ ID NO: 8 in the sequence listing
  • H-271 amino acid sequence represented by SEQ ID NO: 9 in the sequence listing
  • H-296 sequence listing
  • Amino acid sequence represented by SEQ ID NO: 10 CH-271 (amino acid sequence represented by SEQ ID NO: 11 in the sequence listing)
  • CH-296 amino acid sequence represented by SEQ ID NO: 12 in the sequence listing
  • a polypeptide selected from C—CS 1 is exemplified.
  • CH-271, CH-296, C-274, C 1 CS 1 Is a polypeptide having a cell binding domain having an activity of binding to VLA-5.
  • C-CS1, H-296, and CH-296 are polypeptides having a cell-binding domain having an activity of binding to VLA-4.
  • H—27 is a polypeptide having a cell binding domain having an activity of binding to VLA-4.
  • H-296 CH-271 and CH-296 are polypeptides having a heparin-binding domain.
  • fragments in which each of the above domains is modified can also be used.
  • the fibronectin heparin-binding domain has three I I type I sequences (
  • a fragment containing a heparin binding domain lacking one or two of the II type I sequences can also be used in the present invention.
  • CHV-89 an amino acid represented by SEQ ID NO: 14 in the sequence listing
  • fibronectin VLA-5 binding region, Prol 239 to Ser 1515
  • one type III sequence are bound.
  • CHV-90 amino acid sequence represented by SEQ ID NO: 15 in the sequence listing
  • CHV-92 amino acid sequence represented by SEQ ID NO: 16 in the sequence listing
  • two type III sequences Examples of such fragments are CHV-179 (amino acid sequence represented by SEQ ID NO: 17 in the Sequence Listing) and CHV-181 (amino acid sequence represented by SEQ ID NO: 18 in the Sequence Listing).
  • CHV-89, CHV-90, and CHV-92 contain I 1 1-13, 1 1 1-14, III 1-12, respectively
  • CHV-179 contains III-13 and III-14
  • CHV- 181 contains III-12 and III-13, respectively.
  • a fragment obtained by adding an amino acid to each of the above fragments can also be used in the present invention.
  • the fragment can be produced, for example, by adding a desired amino acid to each of the above fragments according to the method for producing H-275-Cys described in the production examples described later.
  • H-275-Cys (amino acid sequence represented by SEQ ID NO: 19 in the sequence listing) is a fragment having a heparin-binding domain of fibronectin and a cysteine residue at the C-terminus.
  • the fragment used in the present invention has the same function as the fragment containing at least a part of the amino acid sequence of natural fibronectin exemplified above as long as the desired effect of the present invention is obtained.
  • polypeptide may be composed of a polypeptide having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions in the amino acid sequence of the polypeptide constituting the.
  • the amino acid substitution and the like are preferably such that the physicochemical properties and the like of the polypeptide can be changed within a range in which the function of the original polypeptide can be maintained.
  • amino acid substitution is a property of the original polypeptide (eg, hydrophobicity)
  • amino acid substitutions are: (1) glycine, alanine; (2) valine, isoleucine, leucine; (3) aspartic acid, glutamic acid, asparagine, glutamine; (4) serine, threonine; (5) lysine, arginine; Amino acid deletions, additions, and insertions are within the range that does not substantially change the properties of the amino acids that are similar to those of the target region of the polypeptide. Deletion, addition, and insertion.
  • a function of maintaining the cytotoxic activity of cytotoxic lymphocytes, a function of enhancing the expression level of IL-12R, or a function of improving the ratio of CD8 positive cells It means having. Whether a fragment comprising a polypeptide having an amino acid substitution or the like has these functions can be appropriately confirmed according to the method described in the Examples below. Further, as a fragment comprising a polypeptide having an amino acid substitution or the like, those having cell adhesion activity and / or heparin binding activity are preferable. Cell adhesion activity and heparin binding activity can be evaluated in accordance with the above-described method for measuring activity.
  • fragment consisting of a polypeptide having amino acid substitution or the like for example, a fragment in which one or more amino acids are inserted as a linker between two different domains. Can also be used in the present invention.
  • fibronectin itself, it is a polypeptide having an amino acid sequence having substitution, deletion, insertion or addition of one or more amino acids in the amino acid sequence of the polypeptide, and at least the above (i) to (iii) Polypeptides having any of these functions can be used in the present invention.
  • the fibronectin fragment described in the present specification can also be produced from a gene recombinant based on the description in US Pat. No. 5,198,423, for example.
  • H-271 SEQ ID NO: 9
  • H-296 SEQ ID NO: 10
  • CH-271 SEQ ID NO: 11
  • CH-296 SEQ ID NO: 12
  • the C-274 (SEQ ID NO: 8) fragment can be obtained by the method described in US Pat. No. 5,102,988.
  • the C—CS 1 (SEQ ID NO: 13) fragment can be obtained by the method described in Japanese Patent No. 3104178.
  • CHV-181 (SEQ ID NO: 18) fragment can be obtained according to the method described in WO 97/18318.
  • CHV-92 (SEQ ID NO: 16) fragment refer to Japanese Patent No. 2729712 and International Publication No. WO 97/18318 pamphlet, and a plasmid was routinely constructed based on the plasmids described in those documents.
  • the plasmid can be used for genetic engineering. .
  • fragments or fragments that can be derived from these fragments, are listed in the following accession numbers at the Patent Microorganism Depositary Center of the National Institute of Advanced Industrial Science and Technology (AIST), 1-chome, 1-chome, Tsukuba, Ibaraki, 305-8566, Japan. Can be produced by using microorganisms deposited under the name, or by modifying plasmids retained by each microorganism by known methods; FERM BP-2264 (Escherichia coli harboring an aramid encoding H-271; deposit date 30 January 1989),
  • FERM BP-2800 Escherichia coli harboring an aramid encoding CH-296; deposit date 12 May 1989
  • FERM BP-2799 Escherichia coli harboring an aramid encoding CH-271; deposit date May 12, 1989
  • FERM BP-7420 E. coli harboring an aramid encoding H-296; deposit date 12 May 1989
  • FERM BP-1915 (a code that codes for C-274. Escherichia coli carrying a lasmid; deposit date 17 June 1988),
  • FERM BP-5723 Erichia coli containing a frasmid that codes for C-CS1; date of deposit March 5, 1990
  • FERM P-12182 Escherichia coli harboring a fluorosmid that codes for CHV-89; deposit date April 8, 1991
  • FERM P-12183 E. coli harboring an aramid encoding CHV-179; date of deposit April 8, 1991. Since fibronectin is a large glycoprotein, it is not always easy to prepare and use proteins of natural origin for industrial and pharmaceutical production. Furthermore, fibronectin is present in large amounts in plasma in vivo, and when fibronectin obtained from plasma is used as a blood product, there is a risk of contamination of components other than fibronectin, which is a safety concern. There may be a problem in In addition, since fibronectin is a multifunctional protein, it can be considered that inconvenience caused by a region different from the region showing the effect of the method of the present invention occurs depending on the situation of its use.
  • a fibronectin fragment and more preferably, a recombinant fibronectin fragment obtained as described above. be able to.
  • the expansion rate of lymphocytes described later is increased, the expression level of IL-2R in expanded lymphocytes is increased, and the proportion of CD8 positive cells in the expanded lymphocyte population is increased.
  • a fibronectin fragment capable of exhibiting the above effects can be particularly preferably used.
  • the molecular weight of the fibronectin fragment used in the present invention is not particularly limited, but is preferably 1 to 200 kD, more preferably 5 to 190 kD, and even more preferably. 1 0 to 1 80 k D.
  • the method of the present invention comprises the production of cytotoxic lymphocytes that perform at least one of induction, maintenance and expansion culture of cytotoxic lymphocytes in the presence of fibronectin, a fragment thereof or a mixture thereof. Is the method.
  • the cytotoxic lymphocyte means a group of cells containing the cytotoxic lymphocyte. In a narrow sense, only cytotoxic lymphocytes contained in the cell group may be indicated.
  • the production of cytotoxic lymphocytes means the induction of cytotoxic lymphocytes from precursor cells that can become the cytotoxic lymphocytes of the present invention, maintenance of cytotoxic lymphocytes, This includes both expansion of cytotoxic lymphocytes using cytotoxic lymphocytes and / or progenitor cells.
  • cytotoxic lymphocytes of the present invention are not particularly limited.
  • CTL cytotoxic T cells
  • LAK cells lymphokine-activated killer cells
  • TIL Tumor infiltrating lymphocytes
  • NK cells NK cells and the like.
  • a progenitor cell that can be a cytotoxic lymphocyte that is, a progenitor cell capable of differentiating into the lymphocyte, includes PBMC, NK cell, naive cell, memory cell, hematopoietic stem cell, cord blood mononuclear cell, etc.
  • PBMC peripheral blood mononuclear cell
  • any blood cell can be used as a progenitor cell in the present invention. These cells can be used either directly or cryopreserved from those collected from a living body.
  • the material containing the cells for example, blood such as peripheral blood and umbilical cord blood, or blood from which components such as red blood cells and plasma are removed, bone marrow A liquid or the like can be used.
  • the method for producing cytotoxic lymphocytes of the present invention comprises cytotoxic lymphocytes in the presence of an active ingredient selected from fibronectin, a fragment thereof or a mixture thereof.
  • an active ingredient selected from fibronectin, a fragment thereof or a mixture thereof.
  • One major feature is the production of spheres.
  • cytotoxic lymphocyte induction, maintenance and expansion or expansion is usually performed in a medium containing a predetermined component in the presence of the active component of the present invention.
  • the number of cells (cytotoxic lymphocytes and / or progenitor cells) used in the present invention is Although not particularly limited, for example, 1 to 1 X 10 8 ce 1 1 s / ml is preferable.
  • the culture conditions are not particularly limited, and the conditions used for normal cell culture can be used. For example, it can be cultured in conditions such as 3 7 ° C, 5 C 0 2. In addition, the medium can be replaced with a fresh one at appropriate time intervals.
  • the medium used in the method for producing cytotoxic lymphocytes of the present invention is not particularly limited, and a known medium prepared by mixing components necessary for maintenance and growth of cytotoxic lymphocytes and their precursor cells.
  • a commercially available medium may be used. These media may contain appropriate proteins, cytokines, and other components in addition to the original components.
  • a medium containing IL-2 is used in the present invention.
  • the concentration of IL-2 in the medium is not particularly limited. For example, it is preferably 0.1 to: LX 10 5 U / ml, more preferably 0.1 to IX 10 4 U / m 1
  • progenitor cells that can become cytotoxic lymphocytes can be co-cultured in a medium further containing an anti-CD3 antibody.
  • concentration of the anti-CD3 antibody in the medium is not particularly limited, but is preferably, for example, 0.01 to 100 g / m1.
  • Anti-CD3 antibody can be added for the purpose of activating the receptor on lymphocytes.
  • lymphocyte stimulating factors such as lectin can also be added.
  • concentration of the component in the medium is not particularly limited as long as a desired effect is obtained.
  • These components can be dissolved in the medium and coexist, or an appropriate solid phase such as Cell culture equipment such as jars, flasks, bags, etc. (including both open and closed systems) or beads, membranes, slide glass, etc. .
  • the material for these solid phases is not particularly limited as long as it can be used for cell culture.
  • the ratio is the same as the desired concentration when the component is dissolved in the medium and used. It is preferable to immobilize a certain amount of each component with respect to the amount of medium to be put in, but the amount of immobilization of the component is not particularly limited as long as a desired effect is obtained.
  • the carrier is used by immersing it in a culture medium in a cell culture device during cell culture.
  • the carrier is placed in the medium, and is added to the equipment so that the concentration is the same as the desired concentration when the component is dissolved in the medium. It is preferable to fix a certain amount of each component with respect to the amount of the medium, but the amount of the component immobilized is not particularly limited as long as a desired effect is obtained.
  • the immobilization of the components can be performed according to a known method, for example, a method for immobilizing a fib mouth nectin fragment described later.
  • acidic polysaccharides acidic oligosaccharides, acidic monosaccharides and salts thereof effective for induction of cytotoxic T cells having antigen-specific cytotoxic activity described in WO 02/14481
  • You may use the compound selected from the group which consists of, and the substance selected from following (A)-(D) with the said component.
  • Examples of the substance having a binding activity to CD 44 include, for example, CD 44 ligand and And / or anti-CD44 antibody.
  • Examples of the substance capable of controlling a signal emitted when a CD44 ligand binds to CD44 include inhibitors of various phosphatases.
  • Examples of substances that can inhibit the growth factor binding to the growth factor receptor include, for example, a growth factor binding activity that inhibits the growth factor from binding to the growth factor receptor by forming a complex with the growth factor. Examples thereof include substances or substances that have binding activity to growth factor receptors and inhibit growth factors from binding to growth factor receptors.
  • examples of the substance capable of controlling the signal emitted by the growth factor binding to the growth factor receptor include inhibitors of various phosphorylases.
  • concentration of these components in the medium is not particularly limited as long as a desired effect is obtained.
  • These components may be dissolved in the medium and coexist, or may be used by being immobilized on an appropriate solid phase as described above.
  • the above various substances may be used alone or in combination of two or more. Can be used.
  • the presence of the active ingredient means that the active ingredient is present in a state capable of exerting its function when performing induction, maintenance or expansion culture of cytotoxic lymphocytes.
  • the content of the active ingredient of the present invention in the medium in which co-culture is performed is not particularly limited as long as a desired effect is obtained. Is from 0.0 1 to 1 0 0 0 0 ⁇ g / m 1, more preferably from 0.1 to 1 00 0 g / m 1, and even more preferably from 1 to 100 g / m 1.
  • the active ingredient is dissolved and coexisted in the medium as described above, and appropriate solid phase, for example, cell culture equipment such as petri dishes, flasks, bags, etc. (open type and closed type) Or a cell culture carrier such as a bead, membrane or slide glass. From the viewpoint of administering cultured cytotoxic lymphocytes to a living body, there is no particular limitation, but it is hoped that the active ingredient is immobilized and used. Good.
  • cytotoxic lymphocytes are obtained by the method of the present invention, and then the lymphocytes and the solid phase are separated.
  • the active ingredient or the like and the lymphocyte can be easily separated, and mixing of the active ingredient or the like into the lymphocyte can be prevented.
  • the ratio is the same as the desired concentration when the ingredient is dissolved in the medium.
  • the active ingredient is immobilized on the carrier, when the carrier is placed in the medium, the amount of medium to be placed in the equipment so that the ratio is the same as the desired concentration when the ingredient is dissolved in the medium.
  • it is preferable to fix a certain amount of each component but it is not particularly limited as long as a desired effect is obtained.
  • immobilization of fibronectin fragments can be carried out by the methods described in WO 97/18318 and FR 00/09168.
  • the expression level of IL-2R is measured for the cytotoxic lymphocytes obtained by the production method of the present invention, at least one of induction, maintenance and expansion culture in the absence of fibronectin, a fragment thereof or a mixture thereof is performed. There is a significant increase in IL-2R expression compared to cytotoxic lymphocytes in which one was performed.
  • the expression level of IL-2R can be measured by a known method, for example, using an anti-IL-2R antibody.
  • the cytotoxic lymphocytes obtained by the method of the present invention have an increased expression level of IL-12R.
  • IL-12R is an activation marker that is expressed on the surface of activated T cells, and with the expression of this molecule, cyto force-in production, cytotoxic activity, proliferation activity, etc. are activated.
  • cytotoxic lymphocytes obtained by the method of the present invention Is a cell group having high functions.
  • the cytotoxic lymphocyte obtained by the method of the present invention has an increased expression level of IL-12R
  • IL-2 added to the medium or a precursor cell of the cytotoxic lymphocyte Increased sensitivity to stimulation by IL-12 produced by lymphocytes themselves or other coexisting cells. For this reason, it can be activated by itself even in an environment with low IL-2 (for example, inside the body).
  • the cytotoxic lymphocytes obtained by the method of the present invention are compared with those obtained by induction, maintenance and expansion culture in the absence of fibronectin, a fragment thereof or a mixture thereof.
  • High percentage of cells with CD8 marker CD8 positive.
  • CD8 positive For example, 1 CD8 positive cells produce cytokines such as interferon and cause immune activation, helper T cell balance becomes Th1 system, 2 CD8 positive cells are cells It is a cell in charge of sex immunity and can efficiently eliminate foreign substances such as viruses and tumor cells.
  • CD8 positive CD8 positive using conventional magnetic beads or flow cytometry Although the cells were purified, the method of the present invention enables the CD8-positive cells to be enriched while culturing.
  • the ratio of CD8-positive cells is large, it can be used as a progenitor cell for inducing CTL. It is suitable for use. (5) Even from a cell population with a low CD8 positive cell ratio, it can be cultured while increasing the CD8 positive cell ratio. Therefore, the method of the present invention is extremely useful in the preparation of cytotoxic lymphocytes.
  • the ratio of CD8 positive cells in the cytotoxic lymphocytes obtained by the method of the present invention is not particularly limited, and can be measured using, for example, an anti-CD8 antibody.
  • cytotoxic lymphocytes prepared by the method of the present invention have a remarkable cytotoxic activity as observed conventionally even if the cultured cells are maintained or proliferated over a long period of time. It has excellent properties such as no degradation The That is, the cytotoxic lymphocytes have higher cytotoxic activity than those induced, maintained and / or expanded in the absence of fibronectin, a fragment thereof or a mixture thereof. It is maintained. Therefore, the cultured cytotoxic lymphocytes can be cloned and maintained as lymphocytes having stable cytotoxic activity. Also induced
  • CTL can be proliferated and expanded by applying antigens, various site strength in, and anti-CD3 antibody stimulation. There are no particular limitations on the maintenance and expansion of the cytotoxic lymphocytes, and known methods can be used.
  • the maintenance of the above-mentioned cytotoxic lymphocyte refers to maintaining the cytotoxic lymphocyte while maintaining the cytotoxic activity.
  • the culture conditions at that time are not particularly limited, and conditions used for normal cell culture can be applied. For example, it can be cultured under conditions such as 37 ° C. and 5% CO 2 .
  • the medium can be replaced with a fresh one at an appropriate time interval.
  • the culture medium used and other components used at the same time are the same as described above.
  • the maintenance and expansion of cytotoxic lymphocytes are carried out by continuously culturing and expanding cytotoxic lymphocytes in the medium in the presence of the active ingredient of the present invention, namely fibronectin, a fragment thereof or a mixture thereof.
  • the active ingredient of the present invention namely fibronectin, a fragment thereof or a mixture thereof.
  • One major feature is culturing. According to expansion culture, the number of cells can be increased while maintaining the cytotoxic activity of cytotoxic lymphocytes. That is, the method of the present invention provides, as one embodiment, a method for expanding cytotoxic lymphocytes.
  • the culture conditions are not particularly limited, and the conditions used for normal cell culture can be used, for example, 37 ° C, 5% C It can be cultured under the conditions of 0 2 etc.
  • the medium can be replaced with a fresh one at an appropriate time interval.
  • the culture medium used and other components used at the same time are the same as described above.
  • the expansion culture method of the present invention for example, in the case of expansion of CTL, CTL having an increased cell number 100 to 1000 times can be obtained by expansion culture for 14 days.
  • the expansion culture of LAK cells LAK cells increased about 200 times by 7 days of culture and 1000 times by 9 days of culture can be obtained.
  • cytotoxic lymphocytes obtained in this way especially CTLs, have higher cytotoxic activity than those obtained by conventional cell lymphocyte expansion culture methods such as the REM method or modified REM method. Holding.
  • Such an effect of the present invention can be confirmed by measuring the cytotoxic activity of CTL or the like expanded by the method of the present invention by the method described in Examples described later.
  • culture can be started from a low number of cells.
  • Adoptive immunotherapy requires a large amount of lymphocytes, but it is difficult to obtain a large amount of lymphocytes from the patient.
  • cell culture equipment in normal expansion of cytotoxic lymphocytes, it is necessary to select cell culture equipment with an appropriate culture area according to the number of cells to be used and culture with an appropriate amount of medium.
  • the amount of cells (number) with respect to the culture area in the cell culture equipment is usually 1 X 10 6 ce 1 1 s / cm 2 or more, Cultivation was started at a high density of 1 X 10 6 cells Zml or higher, and under this cell volume condition, the expansion culture rate [ratio of the number of cells after expansion culture to the number of cells before expansion culture (expansion culture) The number of cells after Z is the number of cells before expansion culture))] becomes very low, and it takes a long culture period to obtain a large amount of cytotoxic lymphocytes.
  • the method of the present invention even when starting with a small amount of cells, Regardless of size, it can be cultured at a high expansion rate. Therefore, the troublesome replacement and dilution operation of cell culture equipment as in the past becomes unnecessary. That is, according to the method of the present invention, sufficient cytotoxic lymphocytes can be expanded by a culture operation using one cell culture device, in other words, by one culture system. Therefore, the method of the present invention is a method for producing cytotoxic lymphocytes that does not include the step of diluting or replacing the cell culture equipment. In particular, with the method of the invention
  • LAK cells When expanding LAK cells, LAK cells can be expanded by adding cells and medium that can become LAK cells to a large volume of cell culture equipment, and then adding IL-12. is there.
  • the present invention is very useful in that a large amount of LAK cells can be obtained by a simple operation.
  • a fibronectin fragment can be preferably used from the viewpoint of obtaining a higher expansion culture rate.
  • a necessary amount of cytotoxic lymphocytes can be obtained in a short time.
  • the culture starts.
  • the amount of cells satisfying the conditions selected from the following (a) and (b) can be used.
  • the ratio of the cell amount to the culture area in the cell culture equipment to be used is preferably 1 to 5 X 10 5 cell sZcm 2 , more preferably 10 to LX 10 5 eel
  • 1 s / cm 2 particularly preferably 1 ⁇ 10 2 to 5 ⁇ 10 4 eells / cm 2 .
  • the concentration of cells in (b) medium preferably l ⁇ 5 X 1 0 5 cell sZml, more preferably 1 0 ⁇ : IX 1 0. 5 ce 1 1 s Zm 1, 1 is particularly preferred X 1 0 2 to 5 X 10 4 ce 1 1 s / m 1.
  • the amount of cells refers to the number of cytotoxic lymphocytes and / or progenitor cells. Further, in the method of the present invention, at least one of induction, maintenance and expansion culture of cytotoxic lymphocytes is performed in one culture system, which does not include steps for cell culture equipment exchange or dilution operation. A method is illustrated.
  • a progenitor cell capable of differentiating into CTL together with an appropriate antigen-presenting cell is added to CTL in the presence of any active medium. It is carried out by incubating (culturing) in.
  • the progenitor cells are not particularly limited as long as they are cells that are pre-staged to become CTL and are destined to differentiate into CTL, such as peripheral blood mononuclear cells (PBMC), naive cells, memory cells, Examples include cord blood mononuclear cells and hematopoietic stem cells.
  • the antigen-presenting cell is not particularly limited as long as it has the ability to present an antigen to be recognized to T cells. For example, a desired antigen can be presented to monocytes, B cells, T cells, macrophages, dendritic cells, fibroblasts, etc., and used in the present invention.
  • the culture conditions of progenitor cells and the like when producing CTL are generally known conditions [for example, Carter J., et al., Immunology, 57 Vol. 1, No. 1, pp. 123-129 (1986)].
  • the medium be suitable for the maintenance and growth of both CTL and feeder cells.
  • a commercially available medium can be used as the medium.
  • the feeder cells used in the method of the present invention are not particularly limited as long as they stimulate CTLs and activate T cell receptors in cooperation with anti-CD3 antibodies.
  • B cells EBV-B cells
  • PBMC PBMC
  • Epstein-Barr virus are used.
  • feeder cells are used after they have been deprived of proliferation ability by means such as irradiation.
  • the content thereof may be determined in accordance with a known method, and for example, 1 X 10 5 to 7 cells / ml is preferable.
  • non-virus-infected cells such as E B V-B cells are used as feeder cells.
  • E B V-B cells are mixed in expanded CTL.
  • Antigen-presenting cells can be prepared by adding an antigenic peptide to cells with antigen-presenting ability and presenting the antigenic peptide on its surface [for example, Bendnarek MA et al. (Bendnarek MA et al.), J Immunol., 147, No. 12, pages 4047-4053 (1991)].
  • a cell having the ability to present an antigen has the ability to process the antigen (proces s)
  • the antigen is taken into the cell and undergoes processing, Fragmented antigenic peptides are presented on the cell surface.
  • an antigen peptide is added to a cell having an antigen-presenting ability, an antigen peptide that matches the MHC restriction property of the antigen-presenting cell to be used and the CTL to be induced is used.
  • the antigen used in the present invention is not particularly limited. Examples thereof include foreign antigens such as bacteria and viruses and endogenous antigens such as tumor-associated antigens (cancer antigens).
  • the antigen-presenting cell is preferably nonproliferative.
  • irradiation with X-rays or treatment with drugs such as mitomycin (mi to myc in) may be performed.
  • progenitor cells capable of differentiating into CTL are incubated (co-cultured) with antigen-presenting cells to induce CTL.
  • General conditions are in accordance with known conditions (for example, see Bendnarek MA et al., J. Immunol., Vol. 147, No. 12, pages 4047-4053 (1991)). Just do it.
  • co-culture conditions can be used conditions used for ordinary cell culture, for example, as possible out culturing under conditions such as 37 ° C, 5% C_ ⁇ 2.
  • This co-culture is usually carried out for about 2 to 15 days, but during that time, antigen-presenting cells may be replaced with newly prepared cells and restimulation may be performed.
  • the medium can be replaced with a fresh one at an appropriate time interval.
  • the CTL obtained by the method of the present invention has an ability to specifically recognize a desired antigen.
  • a cell having the antigen is specifically destroyed by its cytotoxic activity.
  • the cytotoxic activity of CTL can be evaluated by a known method.
  • the cytotoxic activity of CTL against target cells labeled with radioactive substances, fluorescent substances, etc. can be evaluated by measuring the radioactivity and fluorescence intensity derived from target cells destroyed by CTL. It can also be detected by measuring the amount of cytokines such as GM-CSF and IFN-a that are specifically released from CTLs and target cells.
  • it can be directly confirmed by using an antigen peptide-MHC complex labeled with a fluorescent dye or the like.
  • CTL is contacted with a first fluorescent marker force-coupled with a CTL-specific antibody, followed by contact with an antigen-peptide MHC complex force-coupled with a second fluorescent marker, and double labeling.
  • FACS fluorescence-activated cell sorting
  • the CTL expansion culture method of the present invention is not particularly limited as long as the active ingredient is present in the culture system used in the method.
  • the culture The present invention also includes an embodiment in which the active ingredient is present in the system, that is, the precursor cells and the like are cultured in the presence of the active ingredient of the present invention (for example, the active ingredient is added to the medium used for the culture). Included in the invention.
  • the culture of LAK cells is performed by incubating cells that can become LAK cells with IL-2 in the presence of the active ingredient.
  • the cells are not particularly limited, and examples thereof include peripheral blood mononuclear cells (PBMC), NK cells, umbilical cord blood mononuclear cells, hematopoietic stem cells, blood components containing these cells, and the like.
  • LAK cells general conditions for culturing LAK cells are known conditions [for example, cell engineering, Vol. 14, No. 2, p 223-227, (1995); cell culture, 17, (6), pl 92-: 195, (1991); THE LANCET, Vo l. 356, ⁇ 802-807, (2000); Current P rotocolsin Immuno 1 ogy, s up pl e nt 17,
  • co-culture conditions there are no particular limitations on the co-culture conditions, and the conditions used for normal cell culture can be used.
  • the culture can be performed under conditions such as .37 ° C. and 5% CO 2 .
  • This co-culture is usually carried out for about 2 to 15 days.
  • the medium may be replaced with a fresh one at an appropriate time interval.
  • a cell group having high cytotoxic activity for TIL is prepared by culturing in the presence of fibronectin, a fragment thereof, or a mixture thereof.
  • fibronectin, a fragment thereof or a 'mixture thereof is allowed to coexist in the activation operation of these cells.
  • a medium suitable for culturing and activating the cells is used.
  • Appropriate amounts of fibronectin, fragments thereof or mixtures thereof, addition methods, etc. may be selected according to the above methods.
  • the method for producing cytotoxic lymphocytes of the present invention as described above, the cytotoxic activity is maintained high, the expression level of IL-2R is significantly increased, and the ratio of CD8 positive cells is improved. Cytotoxic lymphocytes suitable for use are obtained. Therefore, the method of the present invention, as one aspect thereof, further comprises the step of performing at least one of induction, maintenance and expansion culture of cytotoxic lymphocytes in the presence of the active ingredient of the present invention.
  • a method for improving the ratio of CD8 positive cells in cytotoxic lymphocytes comprising the step of performing at least one of induction, maintenance and expansion culture of cytotoxic lymphocytes in the presence of the active ingredient of the present invention And improving or maintaining cytotoxic activity in cytotoxic lymphocytes, comprising the step of inducing, maintaining and expanding cytotoxic lymphocytes in the presence of the active ingredient of the present invention.
  • an IL-12R expression enhancer on the cell surface containing fibronectin, a fragment thereof or a mixture thereof as an active ingredient.
  • the enhancer may be the active ingredient itself, or any other ingredient such as a medium suitable for the cell to be activated, protein, cytosine (preferably IL-2), other desired Of ingredients.
  • the medium containing the enhancer can be used as a medium for enhancing IL-12R expression in cytotoxic lymphocytes.
  • the medium optionally contains basic components for cell culture.
  • the enhancer and the IL-2R expression enhancing medium can be produced according to known methods using the active ingredient of the present invention.
  • the content of the active ingredient of the present invention in the enhancer or IL-12R expression enhancing medium is not particularly limited as long as the desired effect of the present invention is obtained. According to the content of the active ingredient and the like in the medium used in the method, it can be appropriately determined if desired.
  • the enhancer can enhance the expression of IL-12R on cells in vivo by directly administering to the living body.
  • the present invention contains an agent selected from fibronectin, a fragment thereof, and a mixture thereof as an active ingredient, and a CD 8 positive cell ratio improver in a cultured lymphocyte population Is provided.
  • the ratio improver may be the active ingredient itself, or any other ingredient, for example, a medium suitable for the cell to be activated, protein, cytosine (preferably IL-2), desired It consists of other ingredients.
  • the ratio improver is included.
  • the culture medium can be used as a medium for improving the ratio of CD8 positive cells in cytotoxic lymphocytes.
  • the medium optionally contains basic components for cell culture.
  • the ratio improver and the medium for improving the ratio can be produced according to a known method using the ingredients of the present invention.
  • the ratio improver or the medium for improving the ratio of CD8 positive cells can be appropriately determined as desired in the same manner as in the case of the IL-12R expression enhancer etc.
  • the ratio improver is directly By administering to a living body, the ratio of cytotoxic lymphocytes in the living body can be improved.
  • an agent for improving or maintaining cytotoxic activity in cytotoxic lymphocytes characterized by containing as an active ingredient a component selected from fibronectin, a fragment thereof and a mixture thereof.
  • the enhancer or maintenance agent is the active ingredient itself, or any other optional ingredient, for example, a medium suitable for the cell to be activated, protein, cytosine (preferably IL-1), other desired Of ingredients.
  • the medium containing the enhancer or the maintenance agent can be used as a medium for improving or maintaining the cytotoxic activity in cytotoxic lymphocytes.
  • the medium optionally contains basic components for cell culture.
  • the improver, maintenance agent, improvement medium and maintenance medium can be produced according to a known method using the active ingredient of the present invention.
  • the content of the active ingredient of the present invention in the improver, maintenance agent, enhancement medium and maintenance medium is not particularly limited as long as the desired effect of the present invention is obtained.
  • the method of the present invention According to the content in the medium used in the above, it can be appropriately determined if desired.
  • the activity of the cytotoxic lymphocytes in the living body can be improved or maintained by directly administering the improving agent and the maintaining agent to the living body.
  • the above-mentioned expression enhancer, ratio improver, cytotoxic activity improver and maintainer are solid phase components whose components are appropriate, such as petri dishes, flasks, bags, etc. It may be in the form of material (including both open type and closed type) or a form immobilized on a cell culture carrier such as beads, membranes, and slide glass.
  • lymphocyte-containing culture obtained using the above-described method for producing cytotoxic lymphocytes cells other than cytotoxic lymphocytes such as helper ⁇ cells are usually mixed.
  • the lymphocyte-containing culture obtained by the present invention contains many lymphocytes that retain cytotoxic activity, the cells in the culture are recovered from the culture by centrifugation or the like, The cytotoxic lymphocytes obtained by the method of the present invention can be used as they are. Moreover, if the active ingredient or the like is immobilized on a cell culture device or the like, there is no concern of mixing the ingredient or the like in the obtained cytotoxic lymphocytes.
  • a cell population (or culture) containing the cytotoxic lymphocytes is separated from the culture by a known method and used as the cytotoxic lymphocyte obtained by the method of the present invention. You can also. That is, the method for producing cytotoxic lymphocytes of the present invention can include a step of selecting a cell population having a high content of cytotoxic lymphocytes from the culture obtained by the method.
  • the method of selecting the cell population containing a high amount of cytotoxic lymphocytes For example, an antibody against a cell surface antigen expressed on a desired cell surface from a culture, for example, an anti-CD8 antibody.
  • examples include a method of selectively recovering only the target cells using a cell culture device or carrier to which is bound, and a method of using a flow cytometer.
  • the carrier include magnetic beads and columns.
  • a cell population containing a high amount of target cells can be obtained by adsorbing and removing cells other than the desired cells from the culture.
  • an antibody against a cell surface antigen expressed on the surface of a helper T cell such as an anti-CD4 antibody, can be used to remove the helper T cell from the lymphocyte culture.
  • Flow cytometry can be used for this process. High content of cytotoxic lymphocytes obtained in this way The cell population has a stronger cytotoxic activity than the cell population collected non-selectively from the culture, and can be suitably used particularly in the medical field.
  • the present invention provides cytotoxic lymphocytes obtained by the above-described method for producing cytotoxic lymphocytes of the present invention.
  • the lymphocytes particularly CTL, have a high cytotoxic activity, and have a property that the cytotoxic activity is hardly reduced even if continuous culture or expansion culture is performed for a long period of time.
  • the present invention also provides a medicament (therapeutic agent) containing the lymphocyte as an active ingredient.
  • the therapeutic agent containing the lymphocyte is suitable for use in adoptive immunotherapy.
  • adoptive immunotherapy lymphocytes having cytotoxic activity suitable for patient treatment are administered to the patient, for example, by intravenous administration.
  • the therapeutic agent is according to a method known in the pharmaceutical field.
  • the lymphocyte prepared by the method of the present invention is used as an active ingredient, for example, a known organic or inorganic carrier suitable for parenteral administration. It can be prepared by mixing with excipients, stabilizers and the like.
  • the content of the lymphocyte of the present invention in the therapeutic agent, the dose of the therapeutic agent, and various conditions relating to the therapeutic agent can be appropriately determined according to known adoptive immunotherapy.
  • the method for producing cytotoxic lymphocytes of the present invention can further include a step of introducing a foreign gene into the lymphocytes.
  • the present invention provides, as one aspect thereof, a method for producing cytotoxic lymphocytes, further comprising the step of introducing a foreign gene into the cytotoxic lymphocytes.
  • “Outpatient” refers to being out of the lymphocytes targeted for gene transfer.
  • the method for producing cytotoxic lymphocytes of the present invention particularly the method for expanding cytotoxic lymphocytes, the DNA replication ability of the cultured lymphocytes is enhanced. Therefore, the gene introduction efficiency is expected to increase by including the gene introduction step in the method for producing cytotoxic lymphocytes of the present invention.
  • the gene transfer step can be performed at any time during the production of cytotoxic lymphocytes.
  • the lymphocyte It is preferable from the viewpoint of work efficiency to carry out simultaneously with or after any step of induction, maintenance and / or expansion culture.
  • the virus vector is not particularly limited, and is usually a known virus vector used for gene transfer methods, such as a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a simian virus vector, a vaccinia. Virus vectors or Sendai virus vectors are used. Particularly preferably, the virus vector is retrovirus, adenovirus, adeno-associated virus or simian virus. As the above-mentioned virus vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells.
  • a retroviral vector can stably incorporate a foreign gene inserted into the chromosome DNA of a cell into which the vector is introduced, and is used for gene therapy and the like. Since the vector has a high infection efficiency with respect to dividing and proliferating cells, it is suitable for gene transfer in the production process of cytotoxic lymphocytes in the present invention, for example, in the expansion culture process.
  • the gene transfer method without using a viral vector is not limited to the present invention.
  • a method using a carrier such as a ribosome or a ligand-polylysine, a calcium phosphate method, an electoral position method, a particle gun method, etc. Can be used.
  • a foreign gene incorporated into plasmid DNA or linear DNA is introduced.
  • the foreign gene introduced into the cytotoxic lymphocyte is not particularly limited, and any gene desired to be introduced into the cell can be selected.
  • genes include, for example, proteins (eg, enzymes, cytokinins, In addition to those that encode receptors, etc., antisense nucleic acids and those that encode liposomes can be used.
  • an appropriate marker gene that enables selection of the transfected cell may be introduced at the same time.
  • the foreign gene can be used, for example, by inserting it into a vector or plasmid so that it can be expressed under the control of an appropriate promoter.
  • a promoter and other regulatory elements that cooperate with the transcription start site for example, an enhancer sequence and a terminator sequence, may be present in the vector.
  • homology is made to the nucleotide sequences on both sides of the desired insertion site of the gene in the chromosome.
  • a foreign gene may be placed between the flanking sequences consisting of the nucleotide sequences.
  • the foreign gene to be introduced may be natural or artificially produced or of different origin
  • the DNA molecule may be bound by a known means such as ligation. Furthermore, it may have a sequence in which a mutation is introduced into a natural sequence according to the purpose.
  • a gene encoding an enzyme related to resistance to a drug used for treatment of a patient such as cancer is introduced into cytotoxic lymphocytes, and the drug is added to the lymphocyte. Resistance can be imparted. If such cytotoxic lymphocytes are used, it is possible to combine adoptive immunotherapy and drug therapy, and thus obtain a higher therapeutic effect.
  • cytotoxic lymphocytes are used, it is possible to combine adoptive immunotherapy and drug therapy, and thus obtain a higher therapeutic effect.
  • a multidrug resistance gene is exemplified.
  • a gene that confers sensitivity to a specific drug can be introduced into cytotoxic lymphocytes to impart sensitivity to the drug.
  • lymphocytes after transplantation into a living body can be removed by administration of the drug.
  • An example of a gene that confers sensitivity to a drug is a thymidine kinase gene.
  • Fragment 11-271 derived from human fibronectin was prepared from Escherichia coli HBlOl / pHDlOl (FERM BP-2264) by the method described in US Pat. No. 5,198,423.
  • human fibronectin-derived fragments H-296, CH-271 and CH-296 are Escherichia coli HB101 / Creation 02 (FERM BP-7420), Escherichia coli HBlOl / pCHlOl (FERM BP-2799), Escherichia coli, respectively.
  • HB101 / PCH102 (FERM BP-2800)
  • Fragment derived from human fibronectin ⁇ -274 is Escherichia coli J 109 / pTF7221 (FERM BP-1915), which is cultured by the method described in US Pat. No. 5,102,988, and the culture Prepared from the product.
  • Fragment C-CS1 derived from human fibronectin was cultured from Escherichia coli HB101 / pCS25 (FERM BP-5723) by the method described in Japanese Patent No. 3104178 and prepared from the culture.
  • Fragments CHV-89 and 011-179 derived from human fibronectin use Escherichia coli HB101 / pCHV89 (FERM P-12182) and Escherichia coli HB101 / pCHV179 (FERM P-12183), respectively, and Japanese Patent 2729712 And was prepared from the culture.
  • the fragment CHV-90 derived from human fibronectin is a Japanese patent 27 It was prepared by the method described in the specification of No. 29712. That is, the plasmid PCHV90 was constructed by the operations described in the specification, and the transformant harboring the plasmid was cultured, and CHV-90 was prepared from the culture.
  • Human fibronectin-derived fragment CHV-181 was constructed by constructing a plasmid (PCHV181) containing DNA encoding CHV-181 by the method described in WO 97/18318.
  • E. coli Escherichia coli HB101 / pCHV181 was cultured and prepared from the culture by the same method as C HV-179 described above.
  • the plasmid CHV92 in which the region encoding the III-13 region in the region encoding CHV-181 was deleted was constructed. Deletion operation was performed according to the deletion operation of the III-14 coding region from plasmid pCHV179 described in Japanese Patent No. 2729712.
  • E. coli HB 101 (Escheric hia coli HB101 / pCHV92) transformed with the above plasmid PCHV92 is cultured, and the CHV-89 polypeptide purification method described in Japanese Patent No. 2729712 is used from the culture. Purification operation was performed to obtain a purified CH V-92 standard.
  • Plasmid pCHI02 was prepared from Escherichia coli HB101 / pCH102 (FERM BP-2800). Using this plasmid as a saddle, PCR was performed using primer 12 S, which shows the nucleotide sequence in SEQ ID NO: 20 in the sequence listing, and primer 14 A, which shows the nucleotide sequence in SEQ ID NO: 21 in the sequence listing. An approximately 0.8 kb DNA fragment encoding the binding domain was obtained.
  • the obtained DNA fragment was digested with NcoI and BamHI (both manufactured by Kara Bio Inc.), and then ligated with pTVl 18N (manufactured by Yukari Kara Bio Inc.) digested with Nco I and BamHI. Plasmid pRHl was constructed.
  • the circular DNA obtained in this way was digested with Spe I and Sca'l (manufactured by Yukara Bio) and the 2.3 kb DNA fragment, and the plasmid pRH1-T was transformed into SpeI and Sea A 2.5 kb DNA fragment obtained by digestion with I (manufactured by Kara Bio Inc.) was mixed and ligated to obtain plasmid pRH-Cys.
  • the plasmid was prepared by adding 4-amino acid (Met-8 1 & -8 1 & -3 6 1 ") to the N-terminal side of H-271 and further adding Cys to the C-terminal.
  • the peptide H— 27 5— Cy s is encoded.
  • Polypeptide H-275-Cys was prepared by the following method. Escherichia coli HB101 (Escherichia coli HB101 / pRH-Cys) transformed with the above plasmid pRH-Cys was cultured for 1 ⁇ at 37 X in 120 ml LB medium. Bacterial cells collected from the culture broth were used in 40m 1 destructive buffer (5 OmM Tris_HC and 1 mM. The cells were suspended in EDTA, 15 OmM NaCl, 1 mM DTT, ImM PMSF, pH 7.5), and sonicated to destroy the cells.
  • Escherichia coli HB101 Esscherichia coli HB101 / pRH-Cys transformed with the above plasmid pRH-Cys was cultured for 1 ⁇ at 37 X in 120 ml LB medium. Bacterial cells collected from the culture broth were used in 40m 1 destructive buffer (5 OmM Tris_HC and 1
  • PBMC Middle layer peripheral blood mononuclear cells
  • PBMCs When inducing CTL, these preserved PBMCs are rapidly melted in a 37 ° C water bath, washed with RPMI1640 medium (Bio Whit taker) containing lO ⁇ g / ml DNase (Calbiochem), and then subjected to trypan blue staining. The number of viable cells was calculated and used for each experiment.
  • RPMI1640 medium Bio Whit taker
  • lO ⁇ g / ml DNase Calbiochem
  • non-adherent cells were collected using RPMI1640 medium and stored on ice as responder cells.
  • the isolated monocytes contain 5 g / ml of an influenza virus protein-derived epitope peptide (matrix protein-derived ⁇ 2.1 binding peptide described in SEQ ID NO: 24 in the sequence listing) and lg / ml
  • 5500R X-ray irradiation
  • Peptide solution is aspirated and removed from each well, and the responder cells stored on ice after washing the well with RPMI1640 medium are 0.5-2 ⁇ 10 6 . It was suspended in the 6113/1111 You 51111) 3 ⁇ 411, was added to lm 1 / Ueru Dzu' antigen presenting cells. At this time, each fibronectin fragment described in Production Example 1 (hereinafter referred to as FNfr) was added to a final concentration of 10 g / ml. As a control, a group not added with FNfr was also set. Among plates 5% C_ ⁇ 2, and cultured at 37 ° C.
  • Example 1 The cytotoxic activity of CTL 14 days after the start of induction prepared in (2) was determined by measuring the cytotoxic activity using Cain-AM [Lichtenfel S R., et al. al.), J. Immnol. Methods, Vol.172, No.2, pp.227-239 (1994)].
  • EBV transformed B cells (cell name 221A2..1) co-cultured with or without epotope peptide to give lX10 6 cells / ml
  • Calcein-AM manufactured by Doitite
  • the cells were washed with a medium not containing Calcein-AM and then mixed with 20 times the amount of K562 cells (ATCC CCL-243) to obtain Calcein-labeled target cells.
  • K562 cells were used to eliminate non-specific damage activity by NK cells mixed in responder cells.
  • Example 1 Using memory CTL prepared in (2) as effector cells, serially dilute with 5HRPMI to 1X10 5 to 9X 10 6 eel Is / ml, and then add 100 1 wells to each well of a 96-well cell culture plate. Note that 100 wells of Calcein-labeled target cells prepared to IX 10 5 eel ls / ml were added. After centrifugation for 1 minute The plate containing the above cell suspension 400 x g, incubated for 4 hours at 37 ° C for wet C0 2 incubator within one.
  • the minimum release amount is the release amount of calc ein of the well containing only the target cells and K562 cells, and indicates the spontaneous release amount of calcein from the target cells.
  • the maximum released amount indicates the amount of calcein released when 0.1% of the surfactant Triton X-100 (manufactured by Nacalai Tesque) is added to the target cells to completely destroy the cells.
  • the cytotoxic activity was induced immediately after the induction, but there was almost no difference in the cytotoxic activity depending on the presence or absence of FNfr at the time of induction.
  • Example 1 2 ⁇ 10 5 ceUs CTL prepared in (1) (2) was fixed with PBS (Nissai Tesque) containing 1% paraformaldehyde (Nacalai Tesque), and then washed with PBS. Fixed cells were suspended in 100 I PBS containing 1% BSA (SIGMA). After adding FITC-labeled mouse IgGl or FITC-labeled mouse anti-human CD8 antibody (both from DAK0), 30 Incubated for minutes. After incubation, the cells were washed with PBS and resuspended in PBS containing 1% paraformaldehyde. The cells were subjected to flow cytometry using FACS Vantage (manufactured by Becton Dickinson), and the content of CD8 positive cells was measured. The results are shown in Table 1.
  • Example 1 1 Using the PBMC separated and stored by the method described in (1), anti-influenza virus memory and CTL were induced in the same manner as in Example 1 (2). The cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (1) (3). As a result, there was almost no difference in cytotoxic activity depending on the presence or absence of FNfr at the time of induction.
  • Example 2 Measurement of the expression rate of interorkin-2 receptor (IL-2R) in CTL 14 days after the start of induction prepared in (1) was performed according to the method described in Example 1 (4). went. In this operation, the FITC-labeled mouse anti-human CD8 antibody was changed to a FITC-labeled mouse anti-human IL-12R (CD25) antibody (DAK0). The results are shown in Table 2.
  • Example 1 1 Using the PBMC separated and stored by the method described in (1), anti-influenza virus memory and CTL were induced in the same manner as in Example 1 (2). The cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (1) (3). As a result, there was almost no difference in cytotoxic activity depending on the presence or absence of FNfr at the time of induction.
  • Example 3 The CTL prepared in (1) was washed with 5HRPMI, and then prepared to 3 ⁇ 10 4 eel Is / ml.
  • HLA-A2.1 non-retained allogenic PBMC collected by the same method as in Example 1 (1) was irradiated with X-ray (3300R), washed with medium, and prepared to 2-5X 10 6 eel ls / ml .
  • Cytotoxic activity maintenance (%) was calculated according to the following formula 2.
  • Cytotoxic activity maintenance (%) was calculated according to the following formula 2.
  • CHV- 181 94.5 As shown in Table 3, the CTLs of the various fibronectin fragments added during induction and expansion were more specific after 14 days of expansion compared to controls without the addition of fibronectin fragments. And maintained high cytotoxic activity. In other words, it was clarified that by inducing and expanding in the presence of fibronectin fragment, CTL can be expanded while maintaining high cytotoxic activity for a long time.
  • Example 4 IL-1R expression in cell population after expanded CTL culture
  • Example 1 1 Anti-influenza virus memory CTL was induced in the same manner as in Example 1 1 (2) using PBMCs separated and stored by the method described in (1). The cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (1) (3). As a result, there was almost no difference in cytotoxic activity depending on the presence or absence of FNfr at the time of induction.
  • Example 4- (1) Measurement of Interleukin-1 2 W Expression in Expanded Cultured CTL
  • Example 3- (2) With respect to the CTL after expansion culture thus obtained, the ratio of IL-12R expression positive cells was measured by the method described in Example 2- (2). The results are shown in Table 4.
  • CTL can be expanded while increasing the expression level of IL-2R by inducing and expanding the CTL in the presence of fibronectin fragment.
  • Example 5 CTL induction in the presence of fibronectin, expansion culture
  • Example 1 An anti-influenza virus memory CTL was induced in the same manner as in Example 1_ (2) using PBMC separated and stored by the method described in (1). At this time, fibronectin (manufactured by Calbiochem) was added instead of FNfr so as to have a final concentration of lO g / ml (control was not added). The cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (1) (3), and there was almost no difference in the cytotoxic activity depending on the presence or absence of fibronectin at the time of induction.
  • fibronectin manufactured by Calbiochem
  • Example 5 For the CTL prepared in (1), the ratio of IL-12R expression positive cells was measured by the method described in Example 2- (2). The results are shown in Table 5. Table 5
  • Fibronectin 64.6 As shown in Table 5, CTL induced in the presence of fibronectin showed an increase in IL-12R expression on the cell population.
  • Example 5 Expand the CTL prepared in (1) in the same manner as in Example 3 (2). Nourished. At this time, fibronectin (manufactured by Calbiochem) was added so that fibronectin was added at the time of induction to a final concentration of lO ⁇ g / ral (control was not added). The cytotoxic activity of the obtained CTL was measured by the same method as in Example 1 (1) (3), and “How cytotoxic activity was maintained (%)” was determined as to how much the cytotoxic activity before expansion culture was maintained. Calculated as
  • Fibronectin 148.9 As shown in Table 6, high cytotoxic activity was maintained in the group in which CTL induction and expansion culture were performed in the presence of fibronectin. On the other hand, the cytotoxic activity of the control without fibronectin added during both CTL induction and expanded culture was clearly reduced. In other words, it was clarified that the addition of fibronectin during CTL induction and expansion culture enables the expansion of CTL with specific and long-term cytotoxic activity.
  • Example 6 CTL expansion in the presence of immobilized Fif 'Lonectin (FN) fragment
  • the fibronectin fragment was immobilized on the culture equipment (container) used in the following experiments.
  • the culture equipment used in the following experiments.
  • the plate and flask were washed twice with PBS before use.
  • Example 1 1 Using the PBMC separated and stored by the method described in (1), induction of anti-influenza virus memory and CTL was performed according to the method of Example 1 (1) (2). At this time, a plate in which FNir was immobilized was used as a culture device (a plate not subjected to immobilization treatment was used as a control). When the cytotoxic activity of CTL after induction was evaluated by the method described in Example 1- (3), there was almost no difference in the cytotoxic activity depending on the presence or absence of FNfr immobilization on the plate used for induction.
  • Example 6 The CTL prepared in (2) was expanded according to the method of Example 3 (2). At this time, a flask in which various FNfr were immobilized was used as a culture device (a non-immobilized flask was used for control). In addition, lOHyclone® was used as the medium.
  • CTLs in the group using culture equipment (plates, flasks) with immobilized ragments retained specific and high cytotoxic activity even after expansion.
  • the cytotoxic activity was clearly reduced in the control using equipment that did not immobilize fibronectin fragments during both CTL induction and expansion.
  • CTLs that retain high cytotoxic activity for a long period of time just like fragments dissolved in the medium can be expanded.
  • Example 1 Using the PBMC separated and stored by the method described in (1), the anti-influenza virus memory CTL was induced in the same manner as in Example 1 (2). The cytotoxic activity of CTL on the 14th day after the initiation of induction was evaluated by the method described in Example 1- (3).
  • Example 7 The CTL prepared in (1) was expanded and cultured in the same manner as in Example 3 (2). With respect to the CTL after expansion culture thus obtained, the content ratio of CD8 positive cells was measured by the method described in Example 1 (4). The results are shown in Table 8.
  • Example 6 Culture equipment (container) in the same manner as (1)
  • Example 1 (1) anti-influenza virus memory CTL was induced according to the method of Example 1 (1) (2).
  • the FNfr-immobilized plate prepared in Example 8- (1) was used as a culture device (a plate not subjected to immobilization treatment was used as a control).
  • the cytotoxic activity of CT L was evaluated by the method described in Example 1 '(3). .
  • Example 8 The CTL prepared in (2) was expanded and cultured according to the method of Example 3 (2). At this time, the FNfr-immobilized flask prepared in Example 8- (1) was used as a culture device (a non-immobilized flask was used as a control). In addition, lOHycloneRPMI was used as the culture medium.
  • the ratio of IL-2R expression positive cells was measured by the method described in Example 2- (2).
  • Example 9 Induction of interleukin-1 2 receptor expression on the surface of CD 8 cells (1) Induction of anti-influenza virus memory CTL Example 1 1 Using the PBMC separated and stored by the method described in (1), anti-influenza virus memory and CTL were induced in the same manner as in Example 1 (2).
  • the cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (1) (3). As a result, there was almost no difference in the cytotoxic activity depending on the presence or absence of FNfr at the time of induction.
  • Example 9 The measurement of the expression rate of interleukin-2 receptor (IL-2R) in CTL (especially on the CD 8 cell surface) 14 days after the start of induction prepared in (1) was carried out in Example 1 (4). ). In this operation, FITC-labeled mouse anti-human CD8 antibody was used as the primary antibody, and PE-labeled mouse anti-human IL-12R (CD25) antibody (DAK0) was used as the secondary antibody. The results are shown in Table 10. Table 10
  • Example 1 1 Using the PBMC separated and stored by the method described in (1), anti-influenza virus memory and CTL were induced in the same manner as in Example 1 (2). The cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (3). As a result, there was almost no difference in the cytotoxic activity depending on the presence or absence of fibronectin and FNir at the time of induction.
  • Example 10 The CTL prepared in (1) was expanded and cultured in the same manner as in Example 3 (2). With respect to the CTL obtained before and after the expansion culture, the CD8 positive cell content ratio was measured by the method described in Example 1 (4). The results are shown in Table 11. table 1
  • CD8 positive cell content (%) CD8 positive cell content (%) Control (without FNir) 50. 5 31. 2
  • Example 1 Induction of IL-2R expression in cell populations before and after CTL expansion culture
  • Example 1 1 Anti-influenza virus memory CTL was induced in the same manner as in Example 1 1 (2) using PBMCs separated and stored by the method described in (1). The cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (3). As a result, there was almost no difference in the cytotoxic activity depending on the presence or absence of fibronectin and FNir at the time of induction.
  • Example 11- (1) Measurement of IL-2R Expression Positive Cell Content Ratio in Expanded Cultured CT L CTL prepared in Example 11- (1) was expanded and cultured in the same manner as in Example 3- (2). With respect to the CTL obtained before and after expansion culture, the content ratio of IL-2R expression positive cells was measured by the method described in Example 2- (2). The results are shown in Table 12.
  • Example 1 1 Anti-influenza virus memory CTL was induced in the same manner as in Example 1 1 (2) using PBMCs separated and stored by the method described in (1). The cytotoxic activity of CTL on day 14 after the initiation of induction was evaluated by the method described in Example 1 (3). As a result, there was almost no difference in the cytotoxic activity depending on the presence or absence of fibronectin and FNfr at the time of induction.
  • Example 12 The CTL prepared in (1) was expanded and cultured in the same manner as in Example 3 (2). In addition, lOHyclone RPMI was used as the medium.
  • Table 13 shows the measurement results.
  • the CTL of the group to which fibronectin fragment was added during induction and expansion was unique even after 14 days of expansion compared to the control group to which no fibronectin fragment was added. And possesses high cytotoxic activity. The activity was significantly higher than that of the group to which fibronectin was added.
  • Anti-human CD3 antibody and fibronectin or FN fragment were immobilized on the culture equipment (container) used in the following experiments. That 24-well cell culture plate or 12.5 cm z cell culture flask (manufactured by Falcon) anti-human CD3 antibody (manufactured by Janssen Kyowa Co., Ltd.) (the case of 24-well plates) PBS containing (final concentration 5 g / ml) lml Or 2 ml (in the case of 12.5 cm 2 flask) was added.
  • fibronectin or FN In addition to the fibronectin or each fibronectin fragment (FNfr) described in Production Example 1 at a final concentration of 10 g / ml (for 24-well plates) or 25 g / ml (for 12.5 cm 2 flasks) It added so that it might become. As a control, a group without addition of fibronectin or FNfr was also set.
  • Example 1 (1) Suspend the PBMC prepared in Example 1 (1) in 5HXV I VO 20 to 0.5-lxl0 6 cells / ml, and then immobilize the anti-human CD 3 antibody prepared in Example 13- (1). Plates, or anti-human CD3 antibody and fibronectin or FNfr-immobilized plate were plated at 1 ml / well, and IL-2 (manufactured by Shionogi & Co., Ltd.) was added to a final concentration of 1000 U / ml. These plates were incubated at at 5% C0 2 in 37 (zeroth day of culture).
  • the expansion rate of LAK cells is higher than that in the control group.
  • Example 1 3 LAK cells were induced and cultured in the same manner as in (2). The cell growth rate from the 4th day to the 7th day after the start of the culture was calculated. The results are shown in Table 15. Table 15
  • H-296 43 5-fold As shown in Table 15, in the group using culture equipment with each fibronectin fragment immobilized at the initial stage of LAK cell induction, 4 days from the start of culture compared to the control group. The growth rate of LAK cells from day 7 to day 7 is high. That is, in the early stage of LAK cell induction It was clarified that LAK cells can be induced and cultured at a high growth rate by coexisting with.
  • Example 15 Measurement of expansion rate in LAK cell culture system (Induction / culture of LAK cells from low cell count)
  • Example 13 LAK cells were induced and cultured in the same manner as in (2). This time was set to be the cell concentration at the initiation of culture and 2 X 10 5 ⁇ 1 X 10 6 cells / ml (1 X 10 5 ⁇ 5 X 10 5 C ells / cm 2). On the 15th day after the start of the culture, the number of viable cells was measured by trypan blue staining method, and the expansion culture rate was calculated in comparison with the number of cells at the start of the culture. The results are shown in Table 16.
  • Example 13- (1) antihuman was prepared in the same manner as CD 3 antibody Immobilize plate or anti-human CD 3 antibody and fibronectin or FNfr immobilized 6 well plate, lml / well, add 5HXV I VO20 4ml
  • IL-2 (manufactured by Shionogi & Co., Ltd.) was added to a final concentration of 500 U / ml (lxlO 3 cells / cm 2 ). These plates were incubated at 5% C0 2 in 37 ° C (0 day of culture). IL-2 was added to a final concentration of 500 U / ml on the second, third, and fourth days after the start of the culture. The culture was continued, and IL-2 was added every 2-3 days after the start of the culture every 2-3 days so that the final concentration was 500 U / ml. During this time, the culture broth was not diluted at all.
  • Example 13 LAK cells were induced and cultured in the same manner as in (2). (2) Measurement of IL-2R expression rate in LAK cells
  • Example 17 The IL-2R expression rate in LAK cells induced and cultured in (1) was measured according to the method described in Example 2- (2). The results are shown in Table 18. In such a table 03575
  • Example 13 LAK cells were induced and cultured in the same manner as in (2). (2) Measurement of IL-2R expression rate in LAK cells
  • Example 18 Measurement of IL-2R expression rate on the surface of CD4 cells and CD8 cells in the 7 day LAK cells induced and cultured in (1) according to the method described in Example 9-1 (2) went. In this operation, FITC-labeled mouse anti-human CD 4 antibody or FITC-labeled mouse anti-human CD 8 antibody is used as the primary antibody, and PE-labeled mouse anti-human IL-2R (CD 25) antibody is used as the secondary antibody. used. The results are shown in Table 19. Table 19
  • Fibronectin CD4 / IL-2R double positive cells CD8 / IL-2R 'double positive cells Fragment content (%) Content (%)
  • Example 13 LAK cells were induced and cultured in the same manner as in (2). (2) Measurement of CD8 positive cell population content ratio in LAK cells
  • Example 19 The content ratio of CD8 positive cells in the LAK cells on day 15 induced and cultured in (1) was measured according to the method described in Example 1 (1). The results are shown in Table 20. Table 20
  • Example 1- (1) After suspending 5HXV I V020 to 0.5 ⁇ lxlO s C ells / ml PBMC were made adjustment in Example 1- (1) so that the anti-human CD 3 antibodies immobilized prepared in Example 13- (1) IL-2 (manufactured by Shionogi & Co., Ltd.) was added to the plate or anti-human CD3 antibody and FNfr-immobilized plate in lml / well each so that the final concentration was 1000 U / ml. These Were cultured play Bok in 5% C0 2 in 37 ° C (0 day of culture).
  • an anti-human CD 3 antibody-immobilized flask prepared in the same manner as in Example 13- (1), or an anti-human CD 3 antibody and FNfr-immobilized flask (however, immobilization) (The concentration of the anti-human CD 3 antibody used in the experiment was adjusted to 0.5 g / ml) and the culture medium diluted with 5 H XV I VO 2 0 was appropriately transferred, and IL-2 was added to a final concentration of 500 ⁇ / ⁇ 1 .
  • the culture solution diluted with 5HXV I V020 was transferred again to a new non-immobilized flask, and IL-2 was added to a final concentration of 500 U / mI.
  • the number of viable cells was counted by trypan blue staining method, and calculated as an expansion culture rate compared with the number of cells at the start of culture. The results are shown in Table 21 and Table 22. In the table, donor indicates the symbol of P BMC donor.
  • Example 20 0 LAK cells were induced and cultured in the same manner as in (1). At this time, the cell growth rate from the 4th day to the 8th day after the start of the culture and the cell growth rate from the 1st day to the 1.5th day after the start of the culture were calculated. The results are shown in Tables 23 and 24.
  • Example 20 LAK cells were induced and cultured in the same manner as in (1). (2) Measurement of IL-2R expression rate in LAK cells
  • Example 2 2- Induction in Example 2 2- (1) ⁇ Measurement of IL-2R expression rate in LAK cells on the 15th day after culturing was performed according to the method described in Example 2- (2). The results are shown in Tables 25 and 26. In this table, IL-2R expression positive cell content rate (%) is expressed as IL-2R expression rate (%). Table 2 5
  • Fibronectin Fragment Start of culture 0th day Start of culture Day 9 IL-2R expression rate (%).
  • Example 20 0 LAK cells were induced and cultured in the same manner as in (1).
  • Example 2 3 Measurement of the content ratio of CD8 positive cells in the 15 day LAK cell population induced and cultured in (1) was performed according to the method described in Example 1- (4). The results are shown in Table 27. Table 2 7
  • CD8 positive cell content ratio in the LAK cell population on the 15th day after the start of culture is higher than that in the control group.
  • These CD8 positive cell content ratios were much higher than the CD8 positive cell content ratios in the group using the culture equipment in which only the anti-CD3 antibody was immobilized repeatedly in the early and middle stages of LAK cell induction.
  • stimulation with fibronectin fragments in the early and middle stages of LAK cell induction 0303575
  • LAK cells can be induced and cultured with a high CD8 positive cell content ratio. Sequence listing free text
  • SEQ ID NO: 4 Partial region of f ibronectin named 111-12.
  • SEQ ID NO: 7 Partial region of f ibronectin named CS-1.
  • SEQ ID NO: 24 Designed peptide based on matrix protein derived from inf 1 uenza virus.
  • cytotoxic lymphocytes that maintain high cytotoxic activity, significantly increase the expression level of IL-12R, and improve the ratio of CD8 positive cells.
  • a sphere is obtained.
  • the lymphocyte is suitably used for, for example, adoptive immunotherapy. Therefore, the method of the present invention is expected to make a great contribution to the medical field.

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Abstract

L'invention concerne un procédé de production de lymphocytes cytotoxiques, qui consiste à réaliser au moins l'une quelconque de l'induction, de la maintenance, de la culture dilatatrice des lymphocytes cytotoxiques en présence de la fibronectine, un fragment de celle-ci ou leur mélange.
PCT/JP2003/003575 2002-03-25 2003-03-25 Procede de production de lymphocyte cytotoxique WO2003080817A1 (fr)

Priority Applications (14)

Application Number Priority Date Filing Date Title
AU2003221073A AU2003221073B2 (en) 2002-03-25 2003-03-25 Process for producing cytotoxic lymphocyte
CA2479288A CA2479288C (fr) 2002-03-25 2003-03-25 Procede de production de lymphocyte cytotoxique
KR1020047014928A KR100786054B1 (ko) 2002-03-25 2003-03-25 세포상해성 림프구의 제조방법
MXPA04009287A MXPA04009287A (es) 2002-03-25 2003-03-25 Proceso para producir linfocito citotoxico.
JP2003578546A JP4406566B2 (ja) 2002-03-25 2003-03-25 細胞傷害性リンパ球の製造方法
AT03712887T ATE491021T1 (de) 2002-03-25 2003-03-25 Verfahren zur produktion eines zytotoxischen lymphozyten
EP03712887A EP1496109B1 (fr) 2002-03-25 2003-03-25 Procede de production de lymphocyte cytotoxique
US10/509,055 US8728811B2 (en) 2002-03-25 2003-03-25 Process for producing cytotoxic lymphocyte
DE60335253T DE60335253D1 (de) 2002-03-25 2003-03-25 Verfahren zur produktion eines zytotoxischen lymphozyten
EA200401242A EA010434B1 (ru) 2002-03-25 2003-03-25 Способ получения цитотоксических лимфоцитов
HK05111402.4A HK1079543A1 (en) 2002-03-25 2005-12-12 Process for producing cytotoxic lymphocyte
US11/831,423 US20080227204A1 (en) 2002-03-25 2007-07-31 Process for producing cytotoxic lymphocyte
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US8927273B2 (en) 2003-08-22 2015-01-06 Takara Bio Inc. Process for producing cytotoxic lymphocytes
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US8975070B2 (en) 2002-03-25 2015-03-10 Takara Bio Inc. Process for producing cytotoxic lymphocyte
US8728811B2 (en) 2002-03-25 2014-05-20 Takara Bio Inc. Process for producing cytotoxic lymphocyte
US8927273B2 (en) 2003-08-22 2015-01-06 Takara Bio Inc. Process for producing cytotoxic lymphocytes
AU2005238635B2 (en) * 2004-04-30 2011-11-03 Mabtech Ab Non-elispot assay
US8765469B2 (en) 2005-08-17 2014-07-01 Takara Bio Inc. Method of producing lymphocytes
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WO2007020880A1 (fr) 2005-08-17 2007-02-22 Takara Bio Inc. Procédé de production de lymphocytes
JP4741906B2 (ja) * 2005-08-31 2011-08-10 タカラバイオ株式会社 リンパ球の製造方法
JP2007061020A (ja) * 2005-08-31 2007-03-15 Takara Bio Inc リンパ球の製造方法
WO2007040105A1 (fr) 2005-09-30 2007-04-12 Takara Bio Inc. Procédé de production d'une population de cellules t
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WO2007142300A1 (fr) 2006-06-09 2007-12-13 Takara Bio Inc. Procédé de production de lymphocytes
JPWO2007142300A1 (ja) * 2006-06-09 2009-10-29 タカラバイオ株式会社 リンパ球の製造方法
US8216837B2 (en) 2006-06-09 2012-07-10 Takara Bio Inc. Method of producing lymphocytes
WO2008143014A1 (fr) * 2007-05-11 2008-11-27 Takara Bio Inc. Agent thérapeutique pour le cancer
JPWO2008143014A1 (ja) * 2007-05-11 2010-08-05 タカラバイオ株式会社 がん治療剤
WO2009119793A1 (fr) 2008-03-27 2009-10-01 タカラバイオ株式会社 Procédé de production d'une cellule transfectée
WO2011024791A1 (fr) 2009-08-25 2011-03-03 タカラバイオ株式会社 Procédé de fabrication d'une population de lymphocytes t en présence d'acide rétinoïque
WO2011030851A1 (fr) 2009-09-11 2011-03-17 タカラバイオ株式会社 Procédé de production de cellules tueuses naturelles
US9062287B2 (en) 2009-09-11 2015-06-23 Takara Bio Inc. Process for production of natural killer cells
WO2018135646A1 (fr) 2017-01-20 2018-07-26 国立大学法人京都大学 PROCÉDÉ DE PRODUCTION DE LYMPHOCYTES T CD8α+β+ CYTOTOXIQUES
EP4053268A2 (fr) 2017-01-20 2022-09-07 Kyoto University Procédé de production de lymphocytes t cd8alpha+beta+cytotoxiques
WO2021200901A1 (fr) 2020-03-31 2021-10-07 国立大学法人京都大学 Procédé de production de progéniteurs des lymphocytes t

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AU2008243221A1 (en) 2008-12-04
EP1496109A1 (fr) 2005-01-12
EP2305796B1 (fr) 2012-10-24
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MXPA04009287A (es) 2005-01-25
CA2479288C (fr) 2015-02-24
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ES2356814T3 (es) 2011-04-13
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US20080227204A1 (en) 2008-09-18
ATE491021T1 (de) 2010-12-15
US8728811B2 (en) 2014-05-20
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US8975070B2 (en) 2015-03-10
EP2305796A1 (fr) 2011-04-06

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